LAUSR

Simple Summary CD20 monoclonal antibodies exert a strong ability to quickly kill B cells by using different mechanisms, both direct and indirect, and assessment of killing is difficult. Cytometry emerged as an attractive alternative to classic assays using radioactivity; however, we show here that common cytometry protocols involving centrifugation can lead to missing an important part of the fast killing effect of drugs as mAbs. We show here that rituximab and obinutuzumab direct killing is faster and more important than believed, and that those mAbs differently impact calcium mobilization depending on the time of treatment. We propose here alternative methods to assess fast target cell killing in vitro, avoiding centrifugation or based on survival comparison with control cells or counting beads, both on B cell lines or B-CLL and NHL primary cells. Abstract CD20 monoclonal antibodies (mAbs) eliminate B cells in several clinical contexts. At least two of these Abs, obinutuzumab (OBI) and rituximab (RTX), induce quick elimination of targets and put cancer patients at risk of tumor lysis syndrome (TLS) within 12–24 h of the first dose. The mechanisms of killing can require the recruiting of effector mechanisms from the patient’s immune system, but they can induce direct killing as well. This can be more rapid than recruiting cellular effectors and/or complement. We showed here that OBI and RTX induce quick (<1 h) and high (up to 60% for OBI) killing of two different B cell lines. This was unveiled by using two different techniques that circumvent cell centrifugation steps: a Muse® Cell Analyzer-based approach and a direct examination of the cells’ physical properties by using forward scatter (FS) area and side scatter (SS) area by flow cytometry. These results excluded the presence of aggregates and were also confirmed by developing a normalized survival ratio based on the co-incubation of RTX- and OBI-sensitive cells with MOLM-13, an insensitive cell line. Finally, this normalized survival ratio protocol confirmed the RTX- and OBI-direct killing on primary tumor B cells from B cell chronic lymphocytic leukemia (B-CLL) and Non-Hodgkin’s lymphoma (NHL) patients. Moreover, we unveiled that direct killing is higher than previously expected and absent in patients’ samples at relapse. We also observed that these mAbs, prior to increasing intracellular calcium levels, decrease calcium entry, although manipulating calcium levels did not affect their cytotoxicity. Altogether, our results show that direct killing is a major mechanism to induce cell death by RTX and OBI mAbs.